Microscope suitable for high-throughput screening having an autofocusing apparatus

ABSTRACT

The apparatus for automatically focusing an image in a microscope includes onto an object plane includes an optical system configured to form an optical image of a sample plane to be observed, an autofocusing detection system, and a focus correction system. The autofocusing system includes an autofocusing light beam source for generating autofocusing light beams. The autofocusing system further includes a detection system lens for directing autofocusing light beams to an autofocusing detection device, and an autofocusing detection device for determining the amount of displacement of the image of the object plane from a desired focused reference plane. The focusing correction system includes a feedback controller and focus adjusting device for automatically adjusting the distance between an objective lens and the sample plane in order to properly focus the image in the optical system. A related method of automatically focusing an image of an object plane in a microscope.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a microscope suitable forhigh-throughput screening comprising an autofocusing apparatus having anunfolded, main optical axis. Further the invention also relates to anautofocusing apparatus and to an autofocusing method useful inhigh-throughput screening.

[0003] 2. Description of the Related Art

[0004] Autofocusing techniques for microscopes have been available formany years.

[0005] Most autofocus methods fall into two categories: position sensingand image content analysis. Image content autofocus functions havepreviously been compared for brightfield microscopy. Groen, Young andLigthart (Groen FCA, Young IT, Ligthart G: A comparison of differentfocus functions for use in autofocus algorithms. Cytometry 6:81-91,1985) compared 11 autofocus functions under brightfield using anelectron microscope grid and a metaphase spread, and Vollath (Vollath D:Automatic Focusing by Correlative Methods. J Microsc 147:279-288, 1987)tested an autocorrelation function under brightfield using a paralyticsteel specimen. Groen et al. concluded that three autofocus functions,i.e., two gradient functions and the intensity variance, performed thebest. Its most important limitation is speed, which is dependent on thecalculation performances.

[0006] In a typical autofocusing image content technique, an objectivelens is placed at a predetermined distance from the sample to bescanned, and an image is taken of the object. The image created by themicroscope is then typically evaluated to determine the position atwhich the surface of the object, or a plane within the object, is infocus. The evaluation of the image typically involves analyzingcharacteristics of the image such as entropy, spatial resolution,spatial frequency, contrast, or other characteristics. The analysis ofthese characteristics requires a considerable amount of computerprocessing. Once the characteristics are analyzed, the distance betweenthe objective lens and the object to be scanned is varied, and anotherimage is taken. The new image is then evaluated and the process isrepeated several times before a focused image is finally obtained.Repeating the step of analyzing the image may cause the focusingoperation to take an undesirably long time before the microscope isfinally focused on the object surface. The need for increased processingtime for autofocusing can be particularly acute for various types ofimaging operations. For example, when an object is observed under amicroscope, the focused conditions must be maintained in order tomaintain a properly focused image of the object. Therefore, even if theobject is initially in focus, the object may gradually become out offocus due to a variety of external factors such as thermal effects andvibration, if no corrective steps are taken. Moreover, when an object islarger than the field of view of the microscope, the microscope can onlyfocus on the portion of the object that can be observed through thefield of view of the microscope. Therefore, the focusing conditions mustbe regularly checked and adjusted In order to maintain a sharp image ofthe whole object.

[0007] In view of the foregoing, there is a need for an improvedautofocusing system and method for a microscope that can perform quickand accurate autofocusing operations while maintaining a sharp image.

[0008] The present invention is in a first aspect related to amicroscope having autofocus position sensing means useful inhigh-throughput screening. The uncertainty in applying autofocus testresults from one microscope method to another led to the presentinvention. The development of the present invention included exploringautofocus performance in microscopy of fluorescent stained biologicspecimens.

[0009] Several autofocus position sensing methods and apparatuses areknown, for example from Offenlegungsschrift DE 34 46 727 and DE 33 28821. These German documents disclose an autofocus device for amicroscope wherein variations in light intensity originating from twoseparate light sources provide a signal for focus adjustment. Theseknown autofocus methods are in particular useful for flat samples to beimaged. The autofocusing light beams travel along a substantial largepart including several optical elements, such as a plurality of lensesand at least two beam splitters. This large part causes a substantialdelay in the autofocusing procedure. The present invention is directedto a more simplified autofocus system whereby the part of autofocusinglight is minimized. The uncertainty in applying autofocus test resultsfrom one microscope method to another led to the present invention. Thedevelopment of the present invention included exploring autofocusperformance in microscopy of fluorescent stained biologic specimens.

SUMMARY OF THE INVENTION

[0010] The advantages and purposes of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theadvantages and purposes of the invention will be realized and attainedby means of the elements and combinations particularly pointed out inthe appended claims.

[0011] To attain the advantages and in accordance with the purposes ofthe invention, as embodied and broadly described herein, in a firstaspect the invention is directed toward a microscope for viewing anobject plane. The microscope includes a plurality of lenses positionedalong a main optical axis of the microscope and a probe arm supportingthe plurality of lenses. The probe arm extends generally along the mainoptical axis. The microscope further includes a support on which anobject with an object plane to be examined is placed, the object planesubstantially extending along a focus plane that is observed through themicroscope, and an optical output device for creating an image of theobject plane on an image plane. The main optical axis is unfolded andsubstantially extends along a single plane.

[0012] In yet another aspect, the invention is directed to autofocusingmethods and apparatuses using fluorescent imaging techniques, wherebythe wavelengths of the autofocusing light beams, the illumination andthe excitation light beams are chosen within specified ranges.

[0013] In yet still another aspect, the invention is related to the useof a method for autofocusing in a microscope for high-throughputscreening.

[0014] In another aspect the invention is related to a microscope havingautomatically focusing means for autofocusing an image of an objectplane. The microscope generally includes an optical system configured toform an image of an object plane to be observed, an autofocusingdetection system, and a focusing correction system. The optical systemincludes an objective lens configured to focus on the object plane, anillumination beam source for illuminating the object plane with anillumination light beam, and an image lens configured to create an imageof the object plane. The autofocusing detection system includes anautofocusing light beam source for generating an autofocusing lightbeam, a beamsplitter configured to direct the autofocusing light beam tothe object plane and cause the autofocusing light beam to reflect offthe object plane, a detection system lens configured to direct thereflected autofocusing light beam to an autofocusing detection device,and an autofocusing detection device. The autofocusing detection devicedetermines the amount of displacement of the image of the object planein the optical system from a desired focused reference plane based onthe detected displacement of an image plane of the reflectedautofocusing light beam from a predetermined reference plane in theautofocusing detection system. The autofocusing detection deviceincludes at least one sensor for sensing the reflected autofocusinglight beam and detecting the displacement of the image plane. Thefocusing correction system includes a feedback controller and focusadjusting device for automatically adjusting the distance between theobjective lens and the object plane, based on the reflected autofocusinglight beam sensed by the at least one sensor, in order to properly focusthe image in the optical system.

[0015] In a further aspect, the invention is directed toward a systemfor automatically focusing an image in a microscope. The system includesan imaging system for creating an image of an object plane using anillumination light beam of a first wavelength, and an autofocusingdetection system. The autofocusing detection system includes anautofocusing light beam of a second wavelength. The autofocusing lightbeam is directed to reflect off of the object plane. The autofocusingdetection system further includes a autofocusing detection device havingan iris and a light detector, and a detection system lens. The detectionsystem lens directs the reflected autofocusing light beam to theautofocusing detection device. The autofocusing detection devicereceives the reflected autofocusing light beam from the detection systemlens. The iris permits at least a portion of the reflected autofocusinglight beam to pass through an aperture of the iris. The light detectormeasures the intensity of the portion of the reflected autofocusinglight beam that passes through the aperture of the iris in order todetect the distance that the image of the object plane in the imagingsystem is displaced from a desired focus reference surface.

[0016] In another aspect, the invention is directed toward anotherembodiment of a microscope able to automatically focus an image. Thesystem includes an imaging system for creating an image of an objectplane using an illumination light beam of a first wavelength, and anautofocusing detection system. The autofocusing detection systemincludes an autofocusing light beam of a second wavelength. Theautofocusing light beam is directed to reflect off of the object plane.The autofocusing detection system further includes a autofocusingdetection device comprising a plurality of light sensors, and adetection system lens. The detection system lens directs the reflectedautofocusing light beam to the autofocusing detection device. Theautofocusing detection device receives the reflected autofocusing lightbeam from the detection system lens. The plurality of light detectorsmeasures the light intensity of the reflected autofocusing light beam inorder to detect the distance that the image of the object surface in theimaging system is displaced form a desired focus reference surface.

[0017] In yet another aspect, the invention is directed toward a methodof automatically focusing an image of an object plane in a microscope.The method includes generating an autofocusing light beam, directing theautofocusing light beam against the object plane to be examined, andreflecting the autofocusing light beam off the object plane. The methodfurther includes directing the reflected autofocusing light beam to adetection system and sensing the autofocusing light beam with a lightdetector of the detection system. The method further includesdetermining, based on the sensed autofocusing light beam, the amount ofdisplacement of the image plane of the reflected autofocusing light beamfrom a desired reference plane, and focusing on the object plane tocreate a properly focused image. The sensing includes transmitting thereflected autofocusing light beam at least partially through an apertureof an iris and measuring the light intensity of the reflectedautofocusing light beam that is transmitted through the aperture withthe light detector of the detection system.

[0018] In a further aspect, the invention is directed toward a method ofautomatically focusing an image of an object plane in a microscope. Themethod includes generating an autofocusing light beam, directing theautofocusing light beam against the object plane to be examined, andreflecting the autofocusing light beam off the object plane. The methodfurther includes directing the reflected autofocusing light beam to adetection system and sensing the autofocusing light beam with aplurality of light detectors of the detection system. The method furtherincludes determining, based on the sensed autofocusing light beam, theamount of displacement of the image plane of the reflected autofocusinglight beam from a desired reference plane, and focusing on the objectplane to create a properly focused image. The determining includescomparing the light intensities of the reflected autofocusing light beamdetected by the plurality of light detectors.

[0019] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings, which are incorporated in andconstitute a part of this specification, illustrate several embodimentsof the invention and together with the description, serve to explain theprinciples of the invention. In the drawings,

[0021]FIG. 1 illustrates the basic principles of an optical system forforming an image of an object plane according to the present invention;

[0022]FIG. 2 illustrates the optical system of FIG. 1 with the objectplane in and out of focus;

[0023]FIG. 3A illustrates a microscope with the optical system of FIG. 1and an autofocusing system according to an embodiment of the presentinvention;

[0024]FIG. 3B illustrates an autofocusing detection device of theautofocusing system of FIG. 3A;

[0025]FIG. 3C is a graph of the light intensity detected by theautofocusing detection device of FIG. 3B at various relative positionsbetween an actual image plane and a desired image plane;

[0026]FIG. 4A illustrates a microscope with the optical system of FIG. 1and an autofocusing system according to another embodiment of thepresent invention;

[0027]FIG. 4B illustrates an autofocusing detection device of theautofocusing system of FIG. 4A;

[0028]FIG. 4C is a graph of the intensity of light detected by theautofocusing detection device of FIG. 4B at various relative positionsbetween an actual image plane and a desired image plane;

[0029]FIG. 4D illustrates a variation of the microscope of FIG. 4A witha modified autofocusing detection system;

[0030]FIG. 5 illustrates a microscope with the optical system of FIG. 1and an autofocusing system according to another embodiment of thepresent invention;

[0031]FIGS. 6A, 6B, and 6C illustrate an autofocusing detection deviceof the autofocusing system of FIG. 5 with the object plane in focus, theobject plane too far from an objective lens, and the object plane tooclose to the objective lens, respectively;

[0032]FIGS. 7A, 7B, and 7C illustrate the position of light dots formedon diodes of the autofocusing detection device of FIG. 5 with the objectplane in focus, the object plane too far from the objective lens, andthe object plane too close to the objective lens, respectively;

[0033]FIG. 8A illustrates a microscope with the optical system of FIG. 1and an autofocusing system according to another embodiment of thepresent invention;

[0034]FIGS. 8B, 8C, and 8D illustrate the position of light dots formedon diodes of the autofocusing detection device of FIG. 8A with theobject plane in focus, the object plane too close to the object lens,and the object plane too far from the objective lens, respectively;

[0035]FIG. 9 is an exemplary flowchart of a method of automaticallyfocusing an image of an object plane that can be utilized in themicroscopes of FIGS. 3-4;

[0036]FIG. 10 is an exemplary flowchart of a method of automaticallyfocusing an image of an object plane that can be utilized in themicroscopes of FIGS. 5-8;

[0037]FIG. 11 is a side view of a microscope according to anotherembodiment of the present invention; and

[0038]FIG. 12 is a side view of the microscope of FIG. 11 positioned ona separate table from a scanning stage.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Reference will now be made in detail to the present preferredembodiments of the invention, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same reference numberswill be used throughout the drawings to refer to the same or like parts.

[0040] As of specific importance for a microscope the autofocusingsystems included will be discussed in first instance.

[0041] The present invention provides a microscope having automaticallyfocusing means for automatically focussing the microscope onto a planeof an object such as a sample.

[0042] For reasons of clarity the details of the autofocusing apparatusand system and of the microscope will be discussed separately. However,all features are included.

[0043] According to an embodiment of the invention, an autofocusingapparatus is provided that includes an optical system configured to forman optical image of a sample plane to be observed, an autofocusingdetection system, and a focus correction system. The optical system mayinclude an objective lens, an illumination beam source for illuminatingthe sample plane with an illumination beam, and an image lens, such as aconverging lens, for creating an image of the sample plane. Theautofocusing detection system may include a autofocusing light beamsource for generating an autofocusing light beam, a beamsplitterconfigured to direct the autofocusing light beam to the sample plane andcausing the autofocusing light beam to reflect off the sample surface.

[0044] The autofocusing detection system of the present invention mayfurther include detection system lens configured to direct the returningautofocusing light beam to an autofocusing detection device. Theautofocusing detection device preferably determines the amount ofdisplacement of the image of the sample surface from a desired focusedreference plane based on the detected displacement of an image plane ofthe autofocusing light beam from a predetermined reference plane in theautofocusing detection device. The focusing correction system preferablyincludes a feedback controller and focus adjusting device forautomatically adjusting the distance between the objective lens and thesample plane in order to properly focus the image in the optical system.The present invention also relates to a method of automatically focusingan image of an sample plane in a microscope.

[0045] In a microscope, when the sample plane is not located at thefocal distance from the objective lens, the resulting image in themicroscope will be out of focus. FIGS. 1 and 2, for example, illustratehow this problem can arise in an optical system for forming an opticalimage of a sample to be observed in a microscope. The system of FIGS.1-2 is shown for purposes of illustration only, and does not include theautofocusing system of the present invention which will be described ingreater detail below with reference to FIGS. 3-8.

[0046] As embodied herein and shown in FIGS. 1-2, the optical system 10forms an optical image of a sample plane 16 to be observed in themicroscope. Optical system 10 includes a beamsplitter 12, an objectivelens 14, a partially reflecting object plane or sample plane 16, animage lens 18, an image plane 20, and a source 25 of an illuminationlight beam 22. In the example shown in FIG. 1, the sample plane 16 ofthe sample is positioned at a distance that corresponds to the focaldistance (f1) of the objective lens 14. As a result, in the microscopeof FIG. 1, the resulting image of the sample is properly focused. Incontrast, the sample plane 16 in FIG. 2 is placed at a position thatdeviates (by d1) from the focal distance of the lens 14 and, therefore,the resulting image is not properly focused. In FIGS. 1 and 2, thepartially reflecting plane 16 may correspond to either the bottom of thesample surface or a plane on the inside of the sample. Alternately, thesample plane 16 may correspond to the bottom of the surface on which thesample is placed. For purposes of the discussion below, the plane to befocused will be referred to as the sample plane 16.

[0047] As illustrated in FIGS. 1 and 2, light source 25 generates anillumination light beam 22. The illumination light beam 22 may be of anywavelength that is suitable for illuminating a sample plane in amicroscope. Although the example shown illustrates the illuminationlight beam being collimated, it is not necessary for the beam to becollimated. The beam could also be divergent or convergent. For sake ofdiscussion, the use of a collimated light beam will be described. Thelight source 25 may be any conventional type of light source such as alamp or laser. Although FIGS. 1 and 2 show the light source beinglocated adjacent to a beamsplitter 12, it is also feasible to place thelight source on the left side of the sample plane 16 in the figures inorder to trans-illuminate the sample plane. In such a configuration, thebeam splitter 12 would not be necessary. In an alternate configuration,the sample plane may emit light by itself without the need for aspecific illumination light source. One example of when this may occuris when a sample undergoes a luminescent chemical reaction. The specifictypes of light sources and the preferred wavelengths of the illuminationlight beam will be discussed later with reference to the disclosedautofocusing detection systems of the present invention.

[0048] In the examples of FIGS. 1 and 2, the illumination light beam 22is directed toward a beamsplitter 12. The beamsplitter 12 may be anytype of conventional beamsplitter suitable for use in the presentinvention. The beamsplitter 12 reflects the illumination light beam 22toward the objective lens 14 and sample plane 16 located along the firstoptical axis 56. In FIG. 1, the sample plane 16 is positioned exactly inthe focal plane of the objective lens 14, i.e., at the focal distance f1from the objective lens 14. Because the sample plane 16 is positionedexactly the focal distance away from the objective lens 14, the outerboundaries of the illumination light beam 22 from the objective lens 14will intersect at a single point on the sample plane 16, as shown inFIG. 1. The objective lens 14 is said to be focused on the sample plane16 in such an arrangement where the light beam intersects to strike asingle point on the surface, i.e., the diameter of the light beam willbe a minimum.

[0049] The illumination light beam 22 which strikes the plane 16 isreflected off of the plane 16 and back to the objective lens 14. As theillumination light beam passes through the objective lens 14, theillumination light beam is collimated back into its original form anddirected toward the beamsplitter 12. The beamsplitter 12 is configuredso that the illumination light beam that returns along the first opticalaxis 56 is transmitted through the beamsplitter 12 without anyperturbing effects. After being transmitted through the beamsplitter 12,the collimated light beam reaches the image lens 18.

[0050] The image lens 18 may be any of a variety of conventional lenses,such as a converging lens, for creating an image of a surface. Althoughthe schematic of FIG. 1 only shows an image lens 18, a typicalmicroscope will have a series of relay optics as is known in the art.The relay optics are not shown for purposes of simplicity. In theconfiguration shown in FIG. 1, the image lens 18 projects theillumination light beam onto an image plane 20 positioned at the focaldistance f2 from the image lens 18. A lens such as the image lens 18 (orthe objective lens 14) has a predetermined focal distance (f) based uponits magnification power. In one example of an image lens suitable withthe present invention, the focal distance is between 160 mm to 250 mm.The focal distance of the image lens may be much smaller or larger thanthis range however.

[0051] The focal distance (f) corresponds to the distance from the lensat which a collimated light beam passing through the lens will beproperly focused, i.e., the diameter of the light beam will be at aminimum. For example, the objective lens 14 shown in FIG. 1 has a focaldistance of f1. Therefore, a sample plane 16 located at a distance f1from the objective lens 14 will have the collimated illumination lightbeam 22 from the beamsplitter 12 focused on the sample plane as shown inFIG. 1. Because the sample plane 16 is located at exactly the focaldistance f1 from the objective lens 14, the reflected light beam fromthe surface will be recollimated as it passes back through the objectivelens 14 along the first optical axis 56 toward the image lens 18. Thereflected illumination light beam directed by the image lens 18 (movingto the right in FIG. 1) will then be focused on the image plane 20located at the focal distance f2 (of image lens 18) from the image lens18. Therefore, when the sample plane 16 is at the focal distance f1 fromthe objective lens 14, the resulting image from the image lens 18 willbe properly focused.

[0052] Typically, however, the sample plane 16 is not initiallypositioned at exactly the focal distance from the objective lens 14.Even if the sample plane is initially positioned at the desired distancefrom the objective lens, external factors such as thermal effects orvibrations may cause relative movement between the sample plane and theobjective lens. When the plane 16 is located at position other than thefocal distance f1 away from the objective lens 14 (i.e., moved to theleft or right from the position shown in FIG. 1), the objective lenswill not focus on the sample plane. For example, FIG. 2 shows the sampleplane 16 being located at a greater distance (f1 plus d1) from theobjective lens 14 compared to the distance in FIG. 1. In this newposition, the surface of the sample has moved an additional distance d1from the position shown in FIG. 1. When the sample plane is not locatedat the focal distance f1 from the objective lens, the illumination lightbeam will be focused by the objective lens 14 at the desired referenceplane 23 for the sample (shown in dashed lines in FIG. 2) located f1from the objective lens 14, instead of on the sample plane 16. Thedesired reference plane 23 for the sample (shown in FIG. 2) ispositioned at exactly the focal distance f1 from the objective lens 14,and therefore corresponds to the position at which the diameter of theillumination light beam from the objective lens 14 is at a minimum.

[0053] However, it is desired that the diameter of the illuminationlight beam is at a minimum at the actual plane of sample plane 16 (shownas a solid line), not at a reference plane 23 spaced from the sampleplane. Therefore, it is ultimately desirable for the sample plane 16 tobe placed at the desired reference plane 23 in order for proper focusingto occur. A method and apparatus for obtaining such focusing will bedescribed later.

[0054] In FIG. 2, the sample plane 16 is located an additional distanced1 from the desired reference plane 23 (shown in dashed lines). Thismovement of d1 may be caused by a variety of factors. As seen in FIG. 2,the diameter of the illumination light beam is at a minimum at thedesired reference plane 23 and thus strikes the sample plane 16 at aposition beyond the distance f1 from the objective lens 14. Theillumination beam 22 will then reflect off of the sample plane asreflected light beam 24. Because the illumination light beam 22 did notstrike the sample plane 16 at a single point or minimum diameterposition (as done in FIG. 1), the outer boundaries of the reflectedlight beam 24 will be outside of the outer boundaries of theillumination light beam 22 going toward the sample plane 16. As shown inFIG. 2, after passing back through the objective lens 14, the reflectedlight beams 24 will no longer be collimated.

[0055] The reflected light beams 24 will then transmit through thebeamsplitter 12 toward the image lens 18 along the first optical axis56. Because the reflected light beams are not collimated, the image lens18 will then project the reflected light beams 24 so that they intersectat an image plane 20 which is not at the proper focal distance f2 fromthe image lens 18. The plane located at the focal distance f2 from theimage lens is referred to as the “desired” reference plane for the imageplane. The distance between the desired reference plane 26 for the imageplane (shown in dashed lines) and the actual position of image plane 20(shown as a solid line) is represented as d2 in FIG. 2 and throughoutthe specification. As shown in FIG. 1, when the sample plane is properlypositioned relative to the objective lens, the actual position of theimage plane 20 is identical to the desired reference plane 26.

[0056] An image forming device such as a charge couple device (CCD) orcamera can be positioned at the desired reference plane 26 of theoptical system 10. Alternately, an eyepiece for observing the image maybe positioned at the desired reference plane 26 so that a viewer's eyealigns with the desired reference plane 26. Therefore, in order toproperly focus the optical instrument it is desirable that the reflectedbeam 24 be directed so that the beams intersect at a point on thedesired reference plane 26 (as shown in FIG. 1). For the system shown inFIG. 2, where the sample plane 16 is displaced d1 from the properlyfocused position (a focal distance f1 from the objective lens), therewill be a corresponding displacement d2 of the image plane 20 from thedesired reference plane 26 of the microscope. Therefore, thecorresponding image will be out of focus because it is not at the properfocal distance f2 from the image lens 18.

[0057] Autofocusing systems consistent with the principles of thepresent invention provide quick and accurate automatic focusing onto thesample plane. The autofocusing system includes an autofocusing detectionsystem for directly determining the displacement of the actual imageplane from the desired reference plane of the image plane of the opticalsystem. The displacement generally corresponds to the amount that theimage is out of focus. According to one aspect of the present invention,the need for a time consuming evaluation of the characteristics of aplurality of images is eliminated by directly determining the distancethat the image is out of focus. As a result, the microscope can bequickly and efficiently adjusted so that the image is properly focused.The autofocusing system of the present invention further includes afocusing correction system for adjusting the distance between theobjective lens and the sample plane so that the microscope is quicklyfocused on the sample plane.

[0058] A first example of an apparatus according to the presentinvention for automatically focusing an optical instrument onto a sampleplane is shown in FIGS. 3A, 3B, and 3C. As embodied herein and shown inFIGS. 3A-3C, the apparatus 30 for automatically focusing an opticalinstrument onto a sample plane includes the optical system 10 forforming an image (previously described in FIGS. 1-2), an autofocusingdetection system 32, and a focusing correction system 34. As shown inFIGS. 3A-3C, the apparatus 30 includes an optical system 10 for formingan optical image of a sample to be observed. The optical system 10includes beamsplitter 12, objective lens 14, image lens 18, image plane20, and illumination light source 25 substantially as previouslydescribed in the discussion above for FIGS. 1-2. The principles of theoptical system 10 in FIGS. 3A-3C operate under the same principles aspreviously described for FIGS. 1-2. The components of the optical system10 will be described in greater detail below in relation to theautofocusing detection system 32 and focusing correction system 34.

[0059] As embodied herein and shown in FIGS. 3A-3C, an autofocusingdetection system 32 is provided that includes a light source 39 forgenerating autofocusing light beams 40, a first autofocusingbeamsplitter 42, and a second auto focusing beamsplitter 44. Theautofocusing system 32 further includes a detection system lens 46 fordirecting and returning an autofocusing light beam and a detectiondevice 50 for determining the amount of displacement of the image from adesired focused reference plane. The detection device 50 may be anynumber of devices such as those shown in FIGS. 3A-3C and the otherembodiments of the invention.

[0060] As embodied and shown in FIGS. 3A-3B, light source 39 generatesan autofocusing light beam 40 used in the autofocusing detection system32. The light source 39 may be any suitable light source such as a lampor laser. If a laser is selected, a diode laser or HeNe laser may beused for light sources 39, although any number of other laser types mayalso be used with the present invention. Further, although theautofocusing light beam 40 is shown as being collimated in FIG. 3A, theautofocusing light beam could alternately be either convergent ordivergent. The beam is shown as collimated in order to simplify thediscussion.

[0061] In the example shown in FIG. 3A, the autofocusing light beam hasa wavelength of λa. The wavelength for the autofocusing light beam ispreferably selected to be different than the wavelength of theillumination light beam 52. In most instances, it is preferred that theautofocusing light beam has a longer wavelength than the illuminationlight beam. For the purposes of the description below, the light sourcesand beams will be described in relation to fluorescent imagingspectroscopy, although the present invention is suitable with a largenumber of other applications besides fluorescent imaging spectroscopy.In fluorescent imaging, the illumination light beam 52 has an excitationwavelength of λe and is used for generating the image in the opticalsystem 10, in a manner similar to that discussed with regard to FIG. 1.The wavelengths of the autofocusing light beam 40 and illumination lightbeam 52 are selected to be different from one another so that theautofocusing light beam 40 does not perturb or interfere with theillumination beam 52 used for creating the image.

[0062] In a microscope using fluorescent imaging, the wavelength of theillumination light beam 52 is preferably selected to be as narrow aspossible and within the absorption band of the fluorescent sample understudy. As the illumination light beam strikes the surface, a fluorescentlight beam having a wavelength λf is created. Preferably, the wavelengthof the fluorescent beam is different than the wavelength of theillumination light beam. The difference between the wavelengths, in oneexample, may be as small as 50 nm, preferably 10 nm or smaller. Anylight of the excitation beam should not be allowed to enter the imagelens 18. Therefore, in the example shown in FIG. 3A, the beam splitter12 is configured to block all light with a wavelength λe, while allowinglight of the fluorescence wavelength λf to be transmitted there-through.

[0063] As discussed above, the autofocusing light beams should beselected to have a wavelength (λa) different than the excitationwavelength (λe) and the fluorescent wavelength (λf). One particularexample will be shown for purposes of illustration only. In the case ofa sample that absorbs a wavelength of approximately 510 nm andfluoresces at approximately 550 nm, the excitation beam can be selectedto be an Ar⁺ ion laser with a wavelength of 514 nm. The wavelength ofthe autofocusing light beam can be selected to be greater thanapproximately 600 nm. This one example of the wavelengths is forpurposes of illustration only, and does not limit the present invention.By using different wavelengths, the present system is capable ofsimultaneously determining the amount that the system is out of focusand creating the image of the surface. The ability to perform both ofthese processes simultaneously enhances the speed and efficiency of theautofocusing apparatus.

[0064] In the example shown in FIGS. 3A-3C, the autofocusing light beamsource 39 generates and projects the autofocusing light beam 40 in afirst direction parallel to the first optical axis 56. The autofocusinglight beam 40 strikes the first beamsplitter 42 of the autofocusingdetection system and is reflected along a second optical axis 64 to thesecond beamsplitter 44 of the autofocusing system. Alternately, theapparatus could be configured so that the autofocusing light beam source39 generates the autofocusing light beam 40 directly onto the secondbeamsplitter 44, without needing the first beamsplitter 42. In anotherpossible configuration, the light source 39 for the autofocusing beamcould generate the autofocusing light beam 40 directly to the objectivelens 16.

[0065] The beam splitters 42, 44 used in the present invention may be ofany conventional type known in the art. For example, the beam splitters42, 44 may be partially reflecting conventionally beam splitters. Beamsplitter 44 is preferably configured to transmit all of the illuminationlight beam of wavelengths λe and λf while reflecting the autofocusinglight beams of wavelength λa. In one example, beam splitter 42 ispreferably configured to use a polarizing beam splitter and a ¼wavelength plate. As shown in FIG. 3A, upon striking the secondbeamsplitter 44, the autofocusing light beam 40 is reflected toward theobjective lens 14 along the first optical axis 56. The beamsplitter 44is configured to reflect the autofocusing light beam of wavelength λa.If the beams are operated simultaneously, the beam splitter 44 alsoallows the reflected illumination light beam 52 to pass there-through aspreviously described.

[0066] The autofocusing light beam travels to the objective lens 14along a first optical axis 56. The objective lens 14 may be any type ofmicroscope objective lens. The objective lens 14 has a focal distancef1, which is a function of the magnification power of the objectivelens. For most applications, the effective focal distance f1 willtypically range between 40 mm and 1 mm. However, objective lenses withfocal distances outside of this range are also suitable with the presentinvention. The objective lens 14 directs the autofocusing light beam ofwavelength λa onto the sample plane 16 located at a focal distance f1from the objective lens. In the embodiment shown in FIG. 3A, the sampleplane 16 is located at the focal distance f1 away from the objectivelens, therefore, the resulting image of the sample plane will beproperly focused due to the properties of the optical system (includingthat of the image lens 18).

[0067] The autofocusing light beam 40 from the objective lens 14 is thenat least partially reflected off of the sample plane 16 and directedback to the objective lens 14. The reflected autofocusing light beamwith a wavelength λa is then directed by the objective lens 14 along thefirst optical axis 56 to the second autofocusing beamsplitter 44. Thesecond autofocusing beamsplitter 44 reflects the autofocusing light beamof wavelength λa toward the first autofocusing beamsplitter 42 (in adownward direction along second optical axis 64 in FIG. 3A). The firstautofocusing beamsplitter 42 permits the autofocusing light beamreflected from the second autofocusing beamsplitter 44 to be transmittedthrough without any perturbing effects. The autofocusing light beam 40is thereby transmitted to the detection system lens 46 and autofocusingdetection device 50. The method and apparatus for detecting the amountthat the image is out of focus will be discussed below in greaterdetail.

[0068] As previously discussed, the optical system for creating an imageincludes the source of the illumination light beam for illuminating thesample plane. In a fluorescent imaging system, the illumination lightbeam has a wavelength λe to generate the fluorescence of the sampleplane. As shown in the example of FIG. 3A, the beamsplitter 12 of theoptical system reflects the illumination light beam 52 toward the sampleplane 16 along the first optical axis 56. The second autofocusingbeamsplitter 44 is configured to permit the illumination light beam ofwavelength λe to be transmitted through it to objective lens 14. Theobjective lens 14 then directs the illumination light beam to a point ata distance f1 from the objective lens 14. The illumination light beam isconfigured to intersect at a reference plane corresponding to the focaldistance f1 from the objective lens. In FIG. 3A, because the sampleplane is located f1 from the objective lens, the illumination light beamwill strike a single point on the sample plane and reflect off as shownin FIG. 3A. As the illumination light beam reflects off of the sampleplane it is converted into an illuminated fluorescent beam (in afluorescent imaging example), such as a fluorescent light beam having awavelength of λf. This wavelength is preferably sufficiently differentthan the autofocusing wavelength such that interference does not occurbetween the illuminated beam and the autofocusing light beam.

[0069] The fluorescent light beam from the sample plane 16 has awavelength λf and passes through the objective lens (as it moves to theright in FIG. 3A). In the example shown in FIG. 3A, the fluorescentlight beam is collimated as it passes through the objective lens, anddirected toward the second autofocusing beamsplitter 44. The secondautofocusing beamsplitter 44 is configured to permit the fluorescentlight beam to pass through. The fluorescent light beam then passesthrough the beamsplitter 12 of the image system along the first opticalaxis 56. The image lens 18 then focuses the fluorescent light beam ontoan image plane 20 at a focal distance f2 from the image lens where theimage is formed. In the example shown in FIG. 3A, because the sampleplane 16 is placed exactly the focal distance f1 from the objectivelens, the image plane 20 is coplanar with the desired reference plane 26on which the properly focused image is formed. As previously discussed,the desired reference plane typically corresponds with a surface onwhich may include an image detecting device such as a CCD camera oreyepiece for directly observing the image.

[0070] As previously explained, the present invention incorporates anapparatus and method for directly determining the amount that an imageis out of focus without requiring the analysis of the characteristics ofa plurality of images. The apparatus and method of the present inventiondirectly determines the displacement of the image from its properlyfocused position, and then adjusts the optical system to obtain afocused image. The autofocusing system of the present invention includesan autofocusing detection device for directly determining the amountthat the image is out of focus and includes a focusing correctionsystem.

[0071] The apparatus may include one of several different types ofautofocusing detection devices. FIG. 3A shows an apparatus having oneparticular type of autofocusing detection device according to an aspectof the present invention. The autofocusing detection device 50 of theexample shown in FIG. 3A includes an iris 60 positioned at the focaldistance f3 from the detection system lens 46. The focal distance f3 ofthe detection system lens 46 is a function of the size and magnificationof the detection system lens 46. The iris 60 may be any type of flatplate or other structure with an aperture to permit light to betransmitted there-through.

[0072] When the sample plane 16 is positioned at the proper focusingposition (distance f1 from objective lens 14), the iris will allow theautofocusing light beam of wavelength λa (shown as a solid line in FIG.3B) from the detection system lens 46 to pass through the iris to alight detector 62 without interference. The detection system lens 46preferably has a focal distance f3 that is suitable so that theautofocusing light beam will be small enough to pass through the iriswhen the surface is in focus. The autofocusing light beam will besmaller as the focal length is made larger. However, the autofocusingsystem will be compact and robust with smaller focal distances.Therefore, the selection of the focal distance of the detection systemlens is a balance of these considerations. In one typical embodiment ofthe present invention, the detection system lens 46 has a focal distancebetween 50 mm to 200 mm. The focal distance may be larger or smallerthan this range, however, according to the dimensions and othercharacteristics. In the embodiment shown in FIG. 3A, the light detector62 is positioned on the opposite side of the iris 60 with respect to thedetection system lens 46 along the second optical axis 64.

[0073] During autofocusing, the autofocusing light beam 40 passes thoughthe iris 60 and is transmitted to the detector at a maximum intensitywhen the sample plane 16 is positioned at the distance f1 from theobjective lens. At this position, an image is created at the focaldistance f3 from the detection system lens 46. The image is thus createddirectly on the iris 60 as shown in the solid lines of FIG. 3B. Theintensity of the light measured by the light detector 62 is at its peakvalue because the autofocusing light beam 40 passes substantiallythrough the aperture of the iris 60. At this position, the sample planeis determined to be properly focused by the optical system 10.

[0074] When the sample plane 16 is moved from the position shown in FIG.3A, the autofocusing light beam 40 from the detection system lens 46(shown in dashed lines in FIG. 3B) will not pass directly through theiris without perturbing effects. The beam will intersect at a point “X”positioned a distance d3 from the iris. Thus, for a given displacementd1 of the sample plane 16 from the desired reference plane (for example,the reference plane 23 at a distance f1 from the objective lens 14 asshown in FIGS. 1-2), there will be a corresponding displacement d3 ofthe detection image plane 66 from the plane of the iris 60, as bestshown in FIG. 3B. When the detection image plane 66 is located at adistance from the iris, the intensity of the light measured by the lightdetector is less because not all of the autofocusing light beam willpass through the iris aperture.

[0075]FIG. 3C shows two graphs representing the method used to calculated3 by the light detector 60. The top graph (labeled i) illustrates theintensity of the light (I) measured by the detector versus thedisplacement distance d3. The bottom graph (labeled ii) illustrates thederivative of the intensity of the light (I) measured by the detectorversus the displacement distance d3. As shown by the graphs in FIG. 3C,the light measured by the light detector will be at its maximum when thedistance d3 is zero. Through the measurements of the light detector 62,the displacement distance d2 is determined based on the intensity of thelight beam passing through the iris.

[0076] In the FIGS. 3A and 3B embodiment, it may be difficult todistinguish between a negative and positive d3 (i.e., a beam focusedeither above or below the iris in FIGS. 3A-3C), therefore it ispreferable that the system is modulated in order to solve for thispotential problem. Consequently, the autofocusing detection system ofthe example shown in FIGS. 3A-3C preferably modulates the distance d1with a small amplitude. The modulation results in a change in theintensity of the light, which is proportional to the derivative of theintensity (I). The distance between the sample plane 16 and theobjective lens 14 is preferably adjusted so that the change in intensityis zero, as shown in the bottom graph of FIG. 3C. The value for d3 isthen sent to a feedback controller as will be described below.

[0077] One important aspect of the present invention is that theautofocusing detection system performs the autofocusing based on thecalculated value for the displacement d2 of the image plane 20 from thedesired image plane 26 (see FIGS. 1-2). The autofocusing detectionsystem directly measures the value for d3. The optical system 10 and theautofocusing system 50 may be configured so that a measurement for d3can be directly converted into an value for d2. That is, the value ford2 may be set to be directly related to d3. For example, the lenses ofthe imaging system and autofocusing system may be positioned so that d2is equal to d3. Alternately the lenses may be positioned so that thevalue of d2 is proportional to the value of d3. In another possibleconfiguration, the lenses are positioned so that the value for d2 may bedirectly calculated by an empirical calculation based on d3. In anotherpossible configuration, the value for d2 may be determined based on aset of data points or a map. With each of these options, the measuredvalue for d3 is representative of the value for d2. Therefore, theautofocusing system 50 can detect the amount that the image plane 20 isout of focus without having to analyze the actual characteristics of theimage formed on the image surface. This enhances the speed andefficiency of the autofocusing operation of the present invention. Themethod and structure for focusing the objective lens on the sample planeas a result of the above measurement will be described in greater detailbelow.

[0078] In accordance with present invention, the apparatus includes afocusing correction system 34. As embodied herein and shown in FIGS. 3A,the focusing correction system 34 includes a feedback controller 70 anda focus adjusting device 72. The feedback controller 70 may be an analogor digital feedback controller as is known in the art. The feedbackcontroller 70 receives a signal from the light detector 62 correspondingto the displacement distance d3 and generates a feedback voltage that isthen sent to a focus adjusting device 72.

[0079] The focus adjusting device 72 may be of several different types.In a preferred embodiment, the focus adjusting device 72 adjusts theposition of the objective lens 14 relative to the sample plane 16. Inanother embodiment, the focus adjusting device 72 adjusts the positionof the sample plane 16 relative to the objective lens 14. Either type ofdevice (adjusting the position of the objective lens or adjusting theposition of the sample plane) is designed to position the optical systemso that the sample plane can be quickly put into focus and a focusedimage can be taken of the sample plane. A typical device for impartingthese type of small displacements is a piëzo-positioner. In the exampleshown in FIG. 3A, the focus adjusting device 72 modifies the position ofthe objective lens 14 so that it is at the desired focal distance f1from the sample plane 16. As a result, the image plane 20 of the opticalsystem is placed in focus so that the values for d2 and d3 approachzero. If the autofocusing detection system 50 calculates a value for d3that is above a predetermined threshold, the focusing correction system34 can be operated again until the sample plane is placed in focus. Thisoperation is performed in a shorter period of time because the presentsystem does not analyze the characteristics of the image as is done inother systems.

[0080] Another embodiment of an autofocusing detection device accordingto the present invention is shown in FIGS. 4A, 4B, and 4C. The structureshown in FIGS. 4A and 4B is similar to the example of FIGS. 3A and 3Bexcept for the positioning of the iris. The discussion below willconcentrate on the structure and method that is different than alreadydescribed in relation to FIGS. 3A and 3B. The autofocusing detectiondevice 78 of the example shown in FIGS. 4A-4C includes an iris 80 andlight detector 84. In the autofocusing detection device 78 of FIGS.4A-4C, the iris 80 is placed at a distance not corresponding to thefocal length f3 from the detection system lens 46. That is, the iris isspaced from the reference plane 82 (shown in dashed lines in FIG. 4A),which is located at the focal distance f3 from the detection system lens46. As seen in FIG. 4B, the autofocusing device is designed so that theiris 80 is placed parallel to the reference plane 82 and separated by adistance d3.

[0081] As shown in FIG. 4B, the iris 80 is positioned at a distance off2 minus the distance d3 from the detection system lens 46. In general,in the system of FIGS. 4A-4C, when the light detector 84 measures acertain predetermined intensity, the surface of the sample will be infocus. However, if there are fluctuations in the reflectivity of thesurface or if the power of the light sources fluctuate, the intensitymeasured by the light detector may fluctuate even though the sampleplane is still in focus. The accuracy of the autofocus detection systemthus may be limited by the stability of the light sources and by theuniformity of the sample plane reflectivity. However, even if there arefluctuations in the stability of the light sources or in the surfacereflectivity, the ratio between the light power measured by the detectorand the light source power is not affected by these fluctuations.

[0082] In order to minimize any potential problems due to thesefluctuations, the autofocusing system of the second example may furtherinclude a third autofocusing beamsplitter 90 and a second light detector92, as shown in FIG. 4D. As shown in FIG. 4D, the third autofocusingbeamsplitter 90 is positioned between the detection system lens 46 andthe iris 80 along the second optical axis 64. The second light detector92 is positioned offset from the second optical axis 64 as shown, forexample, in FIG. 4D. The second light detector 92 could also be arrangedat other locations.

[0083] The third autofocusing beamsplitter 90 is configured so that itsplits off a certain percentage of the autofocusing light beam intensityto the second light detector 92, for example, 50%. The intensity (I2) ofthe light split off to the second light detector 92 is proportional tothe total intensity reflected by the plane 16. The remaining 50% of thelight goes to the iris 80. A fraction of this remaining 50% going to theiris 80 is detected by the first light detector 84. The ratio of thelight intensity (I1) detected by the first light detector 84 to thelight intensity (I2) detected by the second light detector 92 is thenused to directly calculate the value d3. By this arrangement,fluctuations in the intensity of the light beams and reflectivity of thesample plane will be accounted for. Alternately, the iris could bereplaced by a diode array positioned where the iris is shown in FIGS. 4Aand 4B. The autofocusing detection system 32 includes a focusingcorrection system 34 similar to that described for FIGS. 3A-3C.

[0084] Another embodiment of an autofocusing detection device accordingto the present invention is shown in FIGS. 5-7. The discussion belowwill concentrate on the structure and method that is different thanalready described in relation to FIGS. 3A and 3B. The autofocusingdetection device 98 of FIGS. 5-7 includes a prism or lens for deflectingthe returning autofocusing light beam of wavelength λa to points on asurface located a distance f3 from the detection system lens. As shownin FIGS. 5-7, a prism 100 is provided between the detection system lens46 and a detection surface 102. The prism 100 deflects the returningautofocusing light beam onto the detection surface 102 located at thefocal distance f3 from detection system lens 46. The focal distance f3shown in FIG. 5 corresponds to the focal distance of the combination ofthe detection system lens 46 and prism 100. Selection of the focaldistance f3 will be discussed below.

[0085] The autofocusing detection system 98 further includes diodepairs. Diode pairs such as 104 and 106 may be positioned on both sidesof the optical axis 64 on the detection surface 102, as shown in FIG. 5.In the example shown in FIGS. 5-7, the first diode pair 104 includes afirst diode 108 and a second diode 110, and the second diode pair 106includes a third diode 112 and a fourth diode 114. In FIGS. 5-7, thefirst diode pair 104 including the first and second diodes 108 and 110are positioned on the left side of the second optical axis 64 as shownin the FIG. 5, and the second diode pair 106 including the third andfourth diodes 112 and 114 are positioned on a right side of the secondoptical axis 64.

[0086]FIGS. 5, 6A, and 7A illustrate aspects of the autofocusingdetection device 98 when the sample plane 16 is located at the focaldistance f1 from the objective lens so that the resulting image of theimage plane 20 is in focus. When the sample plane 16 is properly locatedfor focusing, the autofocusing light beam being directed towards thedetection system lens 46 is typically collimated as shown in FIG. 6A.The detection system lens 46 then projects the autofocusing light beamto prism 100 such as that shown in FIGS. 5 and 6A. In the example shown,the autofocusing light beam is divided by the prism 100 into a firstlight beam 118 and second light beam 120.

[0087] When the plane 16 is properly positioned for focusing, the firstlight beam 118 will focus exactly on the detection surface 102 locatedat the focal distance f3 from the detection system lens 46, as shown inFIG. 5 and 6A. The first light beam 118 will create a first light spot122 halfway between the first diode 108 and the second diode 110, asbest shown in the front view of the diodes in FIG. 7A. The second lightbeam 120 will create a second light spot 124 halfway between the thirddiode 112 and the fourth diode 114, as best shown in FIG. 7A. The lightspots on the diodes will be relatively small because each light beam isat a minimum at the detection surface 102. The sample plane will beproperly focused when the light spots are created as shown in FIG. 7A.

[0088]FIGS. 6B and 7B illustrate aspects of the detection system whenthe sample plane 16 is located more than the focal distance f1 from theobjective lens 14. When the sample plane 16 is located too far from theobjective lens, the returning autofocusing light beam will typically benon-collimated (as shown in FIG. 6B). The detection system lens 46 thenprojects the autofocusing light beam to the prism where the beam isdivided into the first and second light beams 118 and 120, respectively.In the embodiment shown in FIG. 6B, the first light beam 118 is at aminimum diameter and forms an image plane 130 (shown in dashed lines) ata point y1 located at a distance d3 from the detection surface 102 (alsoreferred to as the desired image plane). Because the first light beam118 is not at a minimum diameter at the detection surface 102, the lightspot 122 formed on the diodes is relatively larger than the light spotshown in FIG. 7A. As shown in FIG. 6B and 7B, the second light beam 120is at a minimum diameter at image plane 130 at a point y2 located at thedistance d3 from the detection surface 102. As shown in FIG. 7B, thelight spots 122 and 124 will move inward relative to the light spots ofFIG. 7A.

[0089] When the sample plane is too far from the objective lens asdescribed above, the light spots 122 and 124 are formed primarily on thesecond diode 110 and third diode 112, respectively, as best shown inFIG. 7B. The autofocusing detection system measures the intensity valueat each of the diodes and determines the displacement value d3 of theautofocusing light beam from the reference surface 102. The feedbackcontroller 70 then sends a feedback voltage signal to the focuscorrection system 72 to adjust the distance between the objective lens14 and the sample plane 16 as previously discussed. The plane 16 will beproperly focused when the sum of the intensity measured at the first andfourth diodes is equal to the sum of the intensity measured at thesecond and third diodes.

[0090]FIGS. 6C and 7C illustrate aspects of the autofocusing detectiondevice 98 when the plane 16 is located less than the focal distance f1from the objective lens 14. When the surface is located too close to theobjective lens, the returning autofocusing light beam will alsotypically be non-collimated (as shown in FIG. 6C). In the embodimentshown in FIG. 6C, the first light beam 118 is at a minimum diameter andforms an image plane 132 at a point z1 located at a distance d3 behindthe detection surface 102 (desired image plane). The light spot 122formed on the diodes is relatively larger than the light spot shown inFIG. 7A because light beam 118 is not at a minimum diameter yet when itstrikes the detection surface 102. The second light beam 120 intersectsand forms an image plane at a point z2 also located at the distance d3behind the detection surface 102 as shown in FIG. 7C. As shown in FIG.7C, the light spots 122 and 124 will move outward from the secondoptical axis 64 relative to the light spots of FIG. 7A. The light spots122 and 124 are formed primarily on the first diode 108 and second diode114, respectively, as best shown in FIG. 7C.

[0091] In the arrangement discussed above, the focal distance of thedetection system lens 46 and prism 100 should be selected so that thelight spots 122 and 124 are detectable by the diodes. The light spotsshould be suitably sized so that the diode pairs are able to takeaccurate readings of the light intensity. In one typical diodearrangement, the light spots will be detectable if they have a size ofapproximately 10 μm. Diode arrays with a pixel size of approximately 5μm are known. In applications with fragmented diode arrangements such asshown in FIGS. 5-7 (and FIG. 8), beam displacements of the order of 0.1μm are measurable. This corresponds to an accuracy in the focal distancef1 and positioning of the sample plane of less than 1.0 μm in oneexample of the autofocusing system.

[0092] The focusing correction system 34 of FIGS. 5-7 will function aspreviously described in order to quickly focus the optical system on thesample plane 16 and obtain a focused image.

[0093] Another embodiment of an autofocusing detection device accordingto the present invention is shown in FIGS. 8A-8D. In this example, theautofocusing detection system 108 includes a detection system lenssimilar to that previously described, as well as a cylindrical lens, anda quad photo diode. As embodied herein and shown in FIGS. 8A-8D, acylindrical lens 140 is placed between the detection system lens 46 anda detection surface 142 with a quad photo diode 144. The detectionsurface 142 is preferably located at exactly the focal distance f3 ofthe detection system lens 46. As illustrated in FIG. 8B, the quad photodiode 144 includes first, second, third, and fourth diode segments 146,148, 150, and 152, respectively. The detection system lens 46 andcylindrical lens 140 project the autofocusing light beam onto thedetection surface 142 to form a light spot 154 on the quad diode 144located on the detection surface 142.

[0094] The quad diode 144 determines the displacement d3 of the imagerelative to the detection surface 142 by measuring the light intensityat each of the four diode segments 146, 148, 150, and 152. Thecylindrical lens 140 changes the shape of a light spot 154 depending onthe position of the sample plane relative to the objective lens. FIG. 8Billustrates the position of the light spot 154 when the sample plane 16is properly positioned at the distance f1 from the objective lens 14.The light spot will be located substantially in the center of the fourdiodes. FIG. 8C illustrates the position of the light spot when thesample plane 16 is positioned at a distance less than the focal distancef1 from the objective lens 14. The light spot will be ellipsoidal, withthe majority of the light spot being located on the second diode segment148 and third diode segment 150, as shown in FIG. 8C. Based on themeasured intensity of the diode segments, the feedback controller 70calculates the distance d3 of the image relative to the detectionsurface 142.

[0095]FIG. 8D illustrates the position of the light spot when the sampleplane is positioned at a distance greater than the focal distance f1from the objective lens 14. The light spot will be ellipsoidal, with themajority of the light spot being located on the first diode segment 146and fourth diode segment 152.

[0096] In this example, the feedback controller 70 generates a signal tobe sent to the focus adjusting device for controlling the distancebetween the objective lens 14 and the sample plane 16 in a mannersimilar to that described for the first three examples.

[0097] The autofocusing system of the present invention is suitable fora wide range of applications besides the examples described above. Theselection and arrangement of the light sources depends on the kind ofmicroscopy which is chosen. Although the autofocusing system describedabove is primarily discussed in relation to fluorescent microscopy, thepresent invention is also suitable with other types of microscopy suchas trans-illumination, and luminescence imaging microscopy.

[0098] In trans-illumination microscopy, the source of illumination willenter from the left side of the sample plane in a manner known in theart. The beam splitter 12 as shown in the figures will no longer beneeded. The illumination source may be a lamp with a broad spectrum(i.e., visible spectrum), a lamp filtered by a narrow bandpass filter,or a laser beam. In a trans-illumination system, it may be desirable toadd appropriate filters between beamsplitter 44 and image lens 18 inFIG. 3A. This help prevent any of the autofocusing light beam fromleaking through the beamsplitter 44. When visible light is chosen as theexcitation beam, the light beam of the autofocusing system can be chosenin the infrared range. In one example having a narrow excitationspectrum around 550 nm, a light beam of approximately 633 nm can be usedfor the autofocusing system. These values are shown for purposes ofillustration only.

[0099] In luminescence imaging microscopy, the object emits lightwithout the need of excitation beam. As previously described, a beamsplitter such as beam splitter 12 in FIG. 3A is no longer needed. Thewavelength of the autofocusing light beam is preferably chosen to be farenough from the luminescence wavelength of the sample plane. In such anarrangement, ambient light may assist in the illumination of thesurface. The object itself may be referred to as being the source ofillumination in a luminescence imaging microscopy.

[0100] As previously discussed, a lamp or laser is typically used as theillumination light source. If a lamp is chosen, filters are added toselect the spectrum necessary for the application. If a laser is chosen,the type of laser depends on the wavelength and power needed for thespecific application. Lasers especially suited for the present inventioninclude, for example, Ar⁺ and Kr⁺ lasers. These lasers can typicallyemit light at several discrete wavelengths over the spectrum and arevery versatile for use in a large number of applications. Other types oflaser systems such as an optical parametric oscillator system are alsosuitable with the present invention.

[0101] In all of the autofocusing techniques described above, the sampleplane may be located at either the outside surface of the sample or at aplane on the inside of the sample. In one technique suitable for thepresent invention, the sample surface is used as a reference and thelight beam directed at the sample is offset by a certain amount in orderto scan (or focus) on a plane inside of the sample. This technique isparticularly suited for focusing on the inside of a cell.

[0102] According to another aspect of the invention, a method isprovided for automatically focusing an image of an object plane in amicroscope. Generally, methods consistent with the principles of theinvention include: generating an autofocusing light beam; directing theautofocusing light beam against the object plane to be examined; andreflecting the autofocusing light beam off the object plane. Thereflected autofocusing light beam is then directed to a detectionsystem, where at least one light detector or sensor of the detectionsystem senses the reflected autofocusing light beam. Thereafter, theamount of displacement of the image plane of the reflected autofocusinglight beam from a desired reference plane is determined based on thesensed autofocusing light beam. With this information, the object planecan be focused on to create a properly focused image.

[0103] In methods consistent with the principles of the invention, thestep of sensing may include transmitting the reflected autofocusinglight beam at least partially through an aperture of an iris andmeasuring the light intensity of the reflected autofocusing light beamthat is transmitted through the aperture with the light detector orsensor of the detection system. Alternatively, in methods consistentwith the principles of the invention, the step of determining mayinclude comparing the light intensities of the reflected autofocusinglight beam detected by a plurality of the light detectors or sensors.

[0104] By way of a non-limiting example, FIG. 9 illustrates a methodconsistent with the aspects of the invention for automatically focusingan image of an object plane in a microscope. The embodiment of FIG. 9may be implemented with the autofocusing systems and features discussedin connection with FIGS. 3-4. As illustrated in FIG. 9, an autofocusinglight beam is generated in step 300. For example, the autofocusing lightbeam 40 may be generated by an autofocusing light beam source 39, asshown in FIGS. 3-4. Next, in step 310, the autofocusing light beam isdirected to an object plane, such as object plane 16 in FIGS. 3-4.Thereafter, in step 320, the autofocusing light beam 40 is reflected offthe object plane and directed toward a deflection system such asdetection system 32 in FIG. 3A. The reflected autofocusing light beam isthen transmitted through an iris of the detection system, in step 330.For example, in FIG. 3A, the autofocusing light beam 40 is transmittedthrough iris 60.

[0105] As further shown in FIG. 9, after transmitting the autofocusinglight beam through the iris of the detection system, the autofocusinglight beam is sensed with a light detector or sensor of the detectionsystem, in step 340. To implement this step, a light detector can beselected such as any of the variety of types shown in FIGS. 3-4. Inaddition, to implement step 330 an iris may be provided such as thatshown in FIGS. 3-4. For example, in the embodiment of FIG. 3A, the lightdetector 62 is positioned adjacent the aperture of the iris 60 and theiris is approximately positioned at the focal distance from thedetection system lens 46. Alternatively, as shown in FIG. 4A, the iris80 can be positioned such that it is displaced from the focal distancefrom the detection system lens 46, and a light detector 84 is positionedadjacent the aperture of the iris.

[0106] After sensing the light intensity of the reflected autofocusinglight beam, the amount of displacement of an image plane from a desiredreference plane is determined, as represented in step 350 of FIG. 9.Once again, the features and techniques described above in relation toFIGS. 3-4 may be utilized to determine the amount of displacement of theimage plane based on the sensed, light intensity of the autofocusinglight beam. In step 360, with the determined displacement of the imageplane, the object plane is then focused on in order to create a properlyfocused image. For this purpose, the feedback controller 70 and focusadjusting device 72 of FIGS. 3-4 may be used to adjust the distancebetween the objective lens and the sample or object plane.

[0107]FIG. 10 illustrates another method for automatically focusing animage of an object plane in a microscope. The method of FIG. 10 can beimplemented with the autofocusing systems and features discussed abovein relation to FIGS. 5-8. In the method illustrated in FIG. 10, anautofocusing light beam is generated in step 400. For example, theautofocusing light beam 40 is generated by an autofocusing light beamsource 39, as shown in FIGS. 5-8. In step 410, the autofocusing lightbeam is then directed to an object plane, such as object plane 16.Thereafter, in step 420, the autofocusing light beam 40 is reflected offthe object plane and directed toward a deflection system. As shown atstep 430 in FIG. 10, the reflected autofocusing light beam is thencaused to be sensed by a plurality of light detectors or sensors. Toimplement steps 420 and 430, an arrangement of sensors can be providedas shown in any of the embodiments of FIGS. 5-8. For example, in FIGS.5-7, the reflected autofocusing light beam can be divided into twoseparate light beams 118 and 120 by a prism 100, with the light beamsbeing detected by pairs of light diodes 104 and 106. Alternatively, asshown in FIGS. 8A-8C, the autofocusing light beam can be transmittedthrough a cylindrical lens 140 and sensed by a quad photo diode 144.

[0108] As further shown in FIG. 10, after sensing the reflectedautofocusing light beam with the sensors, the light intensity valuessensed the plurality of sensors are compared at step 440. For example,in FIGS. 5-7, the values of light intensity sensed by each of the fourdiodes 108, 110, 112, and 114 is compared. Alternatively, as shown inFIGS. 8A-8C, the values of the light intensity sensed by each of thefour diode segments 146, 148, 150, and 152 is compared. After the lightintensity values are compared, the amount of displacement of an imageplane from a desired reference plane is determined at step 450. Toimplement this step, the features and techniques described above inrelation to FIGS. 5-8 may be utilized to determine the amount ofdisplacement of the image plane based on the compared values of thesensors. In step 460, with the determined displacement of the imageplane, the object plane is then focused on in order to create a properlyfocused image. For this purpose, the feedback controller 70 and focusadjusting device 72 of FIGS. 5-8 may be used to adjust the distancebetween the objective lens and the object plane.

[0109] The method according the present invention is apparent from theapparatus described above. Other variations may also be made to themethod of the present invention.

[0110] This autofocus method relates in particular to autofocus methodsand apparatus suitable for detecting, characterizing and quantifyingparticulate matter suspended in a fluid. More specifically, theinvention provides an autofocus system for detecting particulates,particularly cells, suspended in a fluid, especially a biological fluid.More in particular, the invention provides an autofocus platform forimaging an affinity-binding based assay.

[0111] Modern drug discovery is limited by the throughput of the assaysthat are used to screen compounds that could possess desired effects. Inparticular, screening of the maximum number of different compoundsnecessitates reducing the time and labor requirements associated witheach screen. In many cases, reaction volumes are very small to accountfor the small amounts of the test compounds that are available.Microscope screening of such small sample volumes compound results inerrors associated with a out of focus images. As these images in generalare the sole measured results further investigation such are computercalculations are performed thereon.

[0112] In high throughput screening tests the speed to obtain andmaintain autofocus is an important factor. In many embodiments, thesamples will be contained in standard multi-well microtiter plates, eachplate having an array of wells, such as those having 96, 384, 1536, orhigher numbers of wells. Standard 96 well microtiter plates which are 86mm by 129 mm, with 6 mm diameter wells on a 9 mm pitch, are used forcompatibility with current automated loading and robotic handlingsystems. Other known microplates are typically 20 mm by 30 mm, with celllocations that are of about 100 to 200 microns in dimension and having apitch of about 500 microns. Both terms ‘well’ and ‘microwell’ usuallyrefer to a specific location in an array of any construction to whichcells adhere and within which the cells are imaged.

[0113] Software procedures can be provided at the user's option to inorder to obtain moves in a Z-axis through a number of differentpositions, acquires an image at each position, and finds the maximum ofa calculated focus that estimates the contrast of each image.

[0114] The present invention is also directed toward an improvedmicroscope comprising an autofocusing apparatus for viewing a sampleplane. The autofocusing apparatus as explained in combination with FIGS.1-10 can be included within or outside the microscope housing. Accordingto the present invention, the microscope includes a plurality of lensespositioned along a main optical axis, a probe arm supporting theplurality of lenses, a support on which a sample plane to be examined isplaced, and an optical output device for creating an image of the sampleplane on an image plane. In the embodiment shown, the main optical axisis unfolded and substantially extends along a single plane. As embodiedherein and shown in FIGS. 11-12, the microscope 200 includes a probe arm213. A series of lenses and other optical devices are positioned alongthe main optical axis 202. In the example shown in FIG. 11, the seriesof lenses includes an emission filter 204, tubus lens 206, illuminationentrance 208, relay lens 210, and corpus lens 212. Any variety ofoptical devices may be positioned along the main optical axis in theprobe arm 213.

[0115] In the example shown in FIG. 11, a plain reflecting mirror 214 ispositioned toward the end of the probe arm closest to the object to bescanned. The surface of the mirror 214 is angled relative to the mainoptical axis 202 so that the light beams along the main optical axis 202may be reflected toward an objective lens 220 and onto the object plane216. In the example shown in FIG. 11, the mirror 214 is angled at, forexample, 45 degrees so that the light from the object plane will bereflected back to the mirror and along the main optical axis 202 to forman image. The sample or object to be examined may be placed directly ona scanning surface 221 corresponding to the object plane 216. The objectsurface may correspond to the surface of the actual object or a planewithin the object. As these objects or samples can have a considerabledepth, the speed of autofocus can further be increased by a volumeimage. Such a volume image can be obtained by observing an image objectat each image plane of a plurality of image planes where each imageplane is vertically displaced with respect to each other image plane.Alternately, the object surface may correspond to the scanning surface221 on which the sample is placed. The samples (or objects) may beplaced in sample holding devices such as one or more microtiter plates218 shown in FIGS. 11 and 12. The microtiter plates 218 and the samplesare supported by a support 219, as shown in FIG. 12. The support 219 ismounted and rigidly secured to a scanning stage 240 that is attached toa first table 242. The support 219 maintains the samples so that theyare physically isolated from the microscope, for reasons which will bediscussed below. The scanning surface 221 may comprise a table movablein a X, X-Y or X-Y-Z motion.

[0116] The microscope of the embodiment shown in FIG. 11 furtherincludes a second plain reflecting mirror 222 at the leftmost portion ofthe main optical axis 202 of the probe arm 213. The light reflected offthe second plain reflecting mirror 222 is directed toward a first videooutput 230 and first video focal plane 232. The microscope also includesa second video output 234 and an eyepiece assembly 236 with an imageplane 238. As is known in the art, the image plane may be either aposition where a viewer's eye is placed, or a place where a camera orimage detecting device is positioned.

[0117] The microscope is preferably configured so that the probe arm 213has an elongated shape as shown in FIGS. 11-12. This is accomplished bydesigning the probe arm so that the main optical axis 202 is unfolded.The elongated shape and unfolded main axis particularly desirable inorder to minimize the transmittal of vibrations from vibrationgenerating structures adjacent the probe arm for reasons which will bediscussed below. The second embodiment of the present invention is alsosuited for microscope devices that scan the image of the object. Duringscanning, scanning motors typically generate undesirable vibrations. Ifthe scanning stage with the scanning motors is placed on or against theprobe arm of the microscope, vibrations from the motors are typicallytransmitted to the microscope, resulting in images of low quality.

[0118] The elongated design of the microscope permits the mounting ofthe microscope on a separate table from the table on which a scanningstage is mounted. For example, as shown in FIG. 12, the scanning stage240 for a microscope may be placed on the first table 242, and themicroscope 200 may be placed on a separate table 244. The first table242 may be a heavy or sturdy table structure, and the scanning stage 240may be rigidly attached or fixed to the surface of the first table. Asfurther shown e in FIG. 12, the microtiter plates 218 may be supportedvia the support structure 219 on the scanning stage 240, so that theplates are physically isolated from the probe arm 213 of the microscope.In this manner, vibrations from the scanning stage 240 are not impartedonto the microscope 200. Therefore, because there are a lack ofvibrations on the microscope, it will be easier to obtain a focusedimage of the sample by either manual or automatic processes.

[0119] In the example shown in FIGS. 11-12, the main optical axis 202 ofthe microscope is configured to be parallel with the object plane 216.This configuration, together with the elongated probe arm, allows theviewer to be positioned at a substantial distance from the samples thatare being observed. This can be particularly desirable if dealing withsamples that involve toxic chemicals, or if the samples must be placedin an isolated room or chamber that is removed from the area in whichthe viewer is located. Moreover, the optics can be changed withouthaving to enter the isolated room or chamber. As a result, the improvedmicroscope of the present invention permits microscopy for a wider rangeof sample types.

[0120] The elongated probe arm and microscope of FIGS. 11-12 also hasother benefits. For example, the longer probe arm allows for scanninglarger objects with the microscope. The longer probe arm also allowsseveral objects to be scanned at once, without unloading and reloadingthe scanning stage. Moreover, the longer probe arm allows the optics tobe more accessible. Because of the design, it is also easier toincorporated additional optics into the probe arm of the instrument. Thelonger probe arm also allows for additional accessories to be included,such as the autofocus system of the first embodiment of the invention.

[0121] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the disclosed embodimentsrelating to an apparatus for automatically focusing an opticalinstrument onto an object plane, a method of automatically focusing anoptical instrument onto an object plane, and a microscope for focusingon an object plane, use of the apparatus of the present invention, andin construction of this apparatus, without departing from the scope orspirit of the invention. The features and aspects of the disclosedembodiments may be combined, modified or substituted to provideadditional advantages and features.

[0122] For example, while features of the invention are disclosed withreference to autofocusing and illumination light beams that aredifferent wavelengths to permit simultaneous operation, it is of coursepossible to select light beams that are of the same or similarwavelength. In such a case, the features of the invention may beimplemented in an asynchronous mode, in which the autofocusing lightbeam is generated and applied at a different time from that of theillumination light beam. It is also possible to implement the lightbeams and features of the invention in an asynchronous mode regardlessof the wavelengths of the light beams (i.e., irrespective of the whetherthe autofocusing light beam and the illumination light beam have thesame or different wavelengths).

[0123] Other embodiments of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered as exemplary only, with a true scope andspirit of the invention being indicated by the following claims.

1. A microscope suitable for high-throughput screening for viewing anobject plane, comprising: a plurality of lenses positioned along a mainoptical axis of the microscope; a probe arm supporting the plurality oflenses, said probe arm extending generally along the main optical axis;a support on which an object with an object plane to be examined isplaced, the object plane substantially extending along a focus planethat is observed through the microscope; an optical output device forcreating an image of the object plane on an image plane, and anautofocusing apparatus for automatically focusing an image of the objectplane, wherein the main optical axis is unfolded and substantiallyextends along a single plane.
 2. The microscope of claim 1, furtherincluding a second optical axis, the second optical axis beingpositioned between the focus plane and the main optical axis, the secondoptical axis being substantially perpendicular to the main optical axis.3. The microscope of claim 2, further comprising a third optical axisbeing positioned between the main optical axis and image plane in theoptical output device, the third optical axis being configured at anangle relative to the main optical axis.
 4. The microscope of any one ofthe claims 1-3, wherein the focusing plane is substantially parallel tothe main optical axis.
 5. The microscope of any one of the claims 1-4,further comprising a scanning stage, said probe arm configured to besubstantially isolated from vibrations created by the scanning stage. 6.The microscope of claim 5, wherein the scanning stage and object arepositioned on a separate support structure than the probe arm of themicroscope, each separate support structure being substantiallyvibrationally isolated from each other.
 7. The microscope of any one ofthe claims 1-6, wherein the object to be examined is positioned on asupport connected to the separate support structure of the scanningstage and said probe arm positioned between the object to be examinedand the scanning stage.
 8. The microscope of any one of the claims 1-7,wherein the probe arm is substantially elongated so that the opticaloutput device may be positioned distant from the object to be examined.9. The microscope according to any one of the claims 1-8, wherein theobject is placed in a sample holding device such as one or more than onemicro titer plates.
 10. The microscope according to any one of theclaims 1-9, wherein the autofocusing apparatus comprises an opticalsystem configured to form an image of an object plane to be observed,said optical system comprising: an objective lens configured to focus onthe object plane, an illumination beam source for illuminating theobject plane with an illumination light beam, and an image lensconfigured to create an image of the object plane; an autofocusingdetection system comprising: an autofocusing light beam source forgenerating an autofocusing light beam, a beamsplitter configured todirect the autofocusing light beam to the object plane and cause theautofocusing light beam to reflect off the object plane, a detectionsystem lens configured to direct the reflected autofocusing light beamto an autofocusing detection device, and an autofocusing detectiondevice for determining the amount of displacement of the image of theobject plane in the optical system from a desired focused referenceplane based on the detected displacement of an image plane of thereflected autofocusing light beam from a predetermined reference planein the autofocusing detection system, said autofocusing detection devicecomprising at least one sensor for sensing the reflected autofocusinglight beam and detecting the displacement of the image plane; and afocusing correction system comprising a feedback controller and focusadjusting device for automatically adjusting the distance between theobjective lens and the object plane, based on the reflected autofocusinglight beam sensed by said at least one sensor, in order to properlyfocus the image in the optical system.
 11. An autofocusing apparatussuitable for high-throughput screening.
 12. The apparatus of claim 11,wherein the autofocusing detection device further comprises an iris forpermitting the reflected autofocusing light beam to pass at leastpartially through an aperture of the iris, said at least one sensormeasuring the intensity of the reflected autofocusing light beam thatpasses through the aperture of the iris.
 13. The apparatus of claim 12,wherein the iris is approximately positioned at the focal distance fromthe detection system lens and wherein the sensor is positioned adjacentthe aperture of the iris.
 14. The apparatus of claim 12, wherein theiris is positioned such that it is displaced from the focal distancefrom the detection system lens and wherein the sensor is positionedadjacent the aperture of the iris.
 15. The apparatus of claim 14,wherein the autofocusing detection device further comprises an auxiliarybeam splitter and an auxiliary light sensor, the auxiliary beam splitterpositioned between the detection system lens and the iris, the auxiliarybeam splitter configured to reflect a fraction of the reflectedautofocusing light beam to the auxiliary light sensor.
 16. The apparatusof claim 15, wherein the displacement of the reflected autofocusinglight beam from the predetermined reference plane is calculated based onthe light intensities measured by the light sensor and auxiliary lightsensor, and wherein the feedback controller calculates the displacementof the image from the desired focused reference plane based on thedisplacement of the reflected autofocusing light beam from apredetermined reference plane.
 17. The apparatus of any one of theclaims 11-16, wherein the at least one sensor comprises a plurality ofdiodes for measuring the light intensity and position of the reflectedautofocusing light beam on a detection surface.
 18. The apparatus ofclaim 17, wherein the autofocusing detection device further comprises aprism positioned between the detection system lens and the plurality ofdiodes, said prism configured to divide the reflected autofocusing lightbeam into at least two separate beams.
 19. The apparatus of claim 18,wherein the plurality of diodes comprise two diode pairs, the firstdiode pair being substantially aligned with a first light beam from theprism, the second diode pair being substantially aligned with a secondlight beam from the prism, said diode pairs measuring the intensity ofthe first and second light beams that strike each diode pair.
 20. Theapparatus of claim 19, wherein the first diode pair is located on afirst side of the optical axis of the detection system lens and thesecond diode pair is located on a second side of the optical axis of thedetection system lens, the first diode pair comprising a first andsecond diode, the second diode pair comprising a third and fourth diode,and wherein the light intensity measured by the individual diodeschanges as a function of the distance between the object plane and theobjective lens.
 21. The apparatus of claim 17, wherein the autofocusingdetection device further comprises a cylindrical lens positioned betweenthe detection system lens and the plurality of diodes, said cylindricallens configured to change the shape of a light spot of the reflectedautofocusing light beam on the plurality of diodes when the distancebetween the object plane and objective lens changes.
 22. The apparatusof claim 21, wherein the plurality of diodes comprises a quad photodiode with four distinct diode segments.
 23. The apparatus of any one ofthe claims 11-22, wherein the feedback controller calculates thedisplacement of the image from the desired focused reference plane basedon the detected displacement of the reflected autofocusing light beamfrom the predetermined reference plane.
 24. The apparatus of one of theclaims 11-23, wherein the autofocusing detection system is configured sothat the measured displacement of the reflected autofocusing light beamfrom the predetermined reference plane is proportional to the amount ofdisplacement of the image from the desired focused reference.
 25. Theapparatus of any one of the claims 11-24, wherein the illumination lightbeam and autofocusing light beam are selected to have differentwavelengths so that the light beams do not interfere with one another.26. The apparatus of any one of the claims 11-25, wherein the focusadjusting device is configured to adjust the position of the objectivelens in order to properly focus the optical system on the object plane.27. The apparatus of any one of the claims 11-26, wherein the focusadjusting device is configured to adjust the position of the objectplane in order to properly focus the optical system on the object plane.28. A system for automatically focusing an image in a microscope,comprising: an imaging system for creating an image of an object planeusing an illumination light beam of a first wavelength; and anautofocusing detection system, said autofocusing detection systemcomprising: an autofocusing light beam of a second wavelength, theautofocusing light beam being directed to reflect off of the objectplane; an autofocusing detection device comprising an iris and a lightdetector; and a detection system lens for directing the reflectedautofocusing light beam to the autofocusing detection device, theautofocusing detection device receiving the reflected autofocusing lightbeam from the detection system lens, said iris permitting at least aportion of the reflected autofocusing light beam to pass through anaperture of said iris, and said light detector measuring the intensityof the portion of the reflected autofocusing light beam that passesthrough the aperture of the iris in order to detect the distance thatthe image of the object plane in the imaging system is displaced from adesired focus reference surface.
 29. The system of claim 28, wherein theiris is approximately positioned at the focal distance from thedetection system lens and the wherein the light detector is positionedadjacent the aperture of the iris.
 30. The system of claim 29, whereinthe iris is positioned such that it is displaced from the focal distancefrom the detection system lens and wherein the light detector ispositioned adjacent the aperture of the iris.
 31. The system of claim30, wherein the autofocusing detection device further comprises anauxiliary beam splitter and an auxiliary light detector, the auxiliarybeam splitter positioned between the detection system lens and the iris,the auxiliary beam splitter configured to reflect a fraction of thereflected autofocusing light beam to the auxiliary light detector. 32.The system of a claim 28, said imaging system comprising an objectivelens, said system further comprising a focus correction systemcomprising a feedback controller and focus adjusting device forautomatically adjusting the distance between the objective lens and theobject plane, based on the reflected autofocusing light beam sensed bysaid light detector, in order to properly focus the image in the imagingsystem.
 33. The system of claim 32, wherein the focus adjusting deviceis configured to adust the position of the objective lens in order toproperly focus the imaging system on the object plane.
 34. The system ofclaim 32, wherein the focus adjusting device is configured to adjust theposition of the object plane lens in order to properly focus the imagingsystem on the object plane.
 35. The system of claim 28, wherein thedistance that the image of the object plane in the imaging system isdisplaced from a desired focus reference surface is a function of thelight intensity measured by the light detector of the autofocusingdetection device.
 36. A system for automatically focusing an image in amicroscope, comprising: an imaging system for creating an image of anobject plane using an illumination light beam of a first wavelength; andan autofocusing detection system, said autofocusing detection systemcomprising: an autofocusing light beam of a second wavelength, theautofocusing light beam being directed to reflect off of the objectplane; an autofocusing detection device comprising a plurality of lightsensors; and a detection system lens for directing the reflectedautofocusing light beam to the autofocusing detection device, theautofocusing detection device receiving the reflected autofocusing lightbeam from the detection system lens, said plurality of light sensorsmeasuring the light intensity of the reflected autofocusing light beamin order to detect the distance that the image of the object plane inthe imaging system is displaced from a desired focus reference surface.37. The system of claim 36, wherein the autofocusing detection devicefurther comprises a prism positioned between the detection system lensand the plurality of light sensors, said prism configured to divide theautofocusing beam into at least two separate beams, the plurality oflight sensors comprising at least two sensor pairs, the first sensorpair being substantially aligned with a first light beam from the prism,the second sensor pair being substantially aligned with a second lightbeam from the prism, said sensor pairs measuring the intensity of thelight beam that strikes each sensor pair.
 38. The system of claim 37,wherein the autofocusing detection device further comprises acylindrical lens positioned between the detection system lens and theplurality of light sensors, said cylindrical lens configured to changethe shape of a light spot on the plurality of diodes when the distancebetween the object plane and objective lens changes.
 39. The system ofclaim 38, wherein the plurality of light sensors comprises a quad photodiode with four distinct diode segments.
 40. The system of a claim 36,said imaging system comprising an objective lens, said system furthercomprising a focus correction system comprising a feedback controllerand focus adjusting device for automatically adjusting the distancebetween the objective lens and the object plane, based on the reflectedautofocusing light beam sensed by said light detector, in order toproperly focus the image in the imaging system.
 41. The system of claim40, wherein the focus adjusting device is configured to adjust theposition of the objective lens in order to properly focus the imagingsystem on the object plane.
 42. A method of automatically focusing animage of an object plane in a microscope, comprising: generating anautofocusing light beam; directing the autofocusing light beam againstthe object plane to be examined; reflecting the autofocusing light beamoff the object plane; directing the reflected autofocusing light beam toa detection system; sensing the autofocusing light beam with a lightdetector of the detection system; determining, based on the sensedautofocusing light beam, the amount of displacement of the image planeof the reflected autofocusing light beam from a desired reference plane;and focusing on the object plane to create a properly focused image,wherein said sensing includes transmitting the reflected autofocusinglight beam at least partially through an aperture of an iris andmeasuring the light intensity of the reflected autofocusing light beamthat is transmitted through the aperture with the light detector of thedetection system.
 43. The method of claim 42 comprising an autofocusingapparatus according to any of the claims 11-27 and a system according toany of the claims 28-41.
 44. The method of claim 42, wherein the iris isapproximately positioned at the focal distance from a detection systemlens and wherein the light detector is positioned adjacent the apertureof the iris.
 45. The method of claim 42, wherein the iris is positionedsuch that it is displaced from the focal distance from a detectionsystem lens and wherein the light detector is positioned adjacent theaperture of the iris.
 46. The method of claim 45, wherein said directingincludes reflecting a fraction of the autofocusing light beam via a beamsplitter to a second light detector and measuring the light intensity atthe second light detector.
 47. The method of claim 42, furthercomprising, simultaneously with said generating of the autofocusinglight beam: generating an illumination light beam; illuminating theobject plane with the illumination light beam; and reflecting theillumination light beam off the object plane to create an image of theobject plane.
 48. The method of claim 47, wherein said focusing includescreating a reference signal representative of the amount of displacementof the image of the object plane from a desired focused reference plane.49. A method of automatically focusing an image of an object plane in amicroscope, comprising: generating an autofocusing light beam; directingthe autofocusing light beam against the object plane to be examined;reflecting the autofocusing light beam off the object plane; directingthe reflected autofocusing light beam to a detection system; sensing theautofocusing light beam with a plurality of light detectors of thedetection system; determining, based on the sensed autofocusing lightbeam, the amount of displacement of the image plane of the reflectedautofocusing light beam from a desired reference plane; and focusing onthe object plane to create a properly focused image, wherein saiddetermining includes comparing the light intensities of the reflectedautofocusing light beam detected by the light detectors.
 50. The methodof claim 49, wherein said sensing the autofocusing light beam includesdividing the autofocusing light beam into at least two separate lightbeams by a prism positioned between a detection system lens and theplurality of light detectors.
 51. The method of claim 50, wherein saidsensing the autofocusing light beam includes measuring the lightintensity at two diode pairs, the first diode pair being substantiallyaligned with a first light beam from the prism, the second diode pairbeing substantially aligned with a second light beam from the prism. 52.The method of claim 49, wherein said sensing the autofocusing light beamincludes transmitting the autofocusing light beam through a cylindricallens positioned between a detection system lens and the plurality oflight detectors to alter the shape of a light beam projected onto thelight detectors.
 53. The method of claim 52, further comprisingproviding, as the light detectors, a quad photo diode with four distinctdiode segments.
 54. The method of claim 49, further comprising,simultaneously with generating the autofocusing light beam: generatingan illumination light beam; illuminating the object plane with theillumination light beam; and reflecting the illumination light beam offthe object plane to create an image of the object plane.
 55. The methodof claim 54, wherein said focusing includes creating a reference signalrepresentative of the amount of displacement of the image of the objectplane from a desired focused reference plane.